Bile acid is synthesized in the liver and secreted into a small intestine, and plays an important role in promoting absorption of lipids, lipid-soluble vitamins and cholesterols in the small intestine. Bile acid is re-absorbed efficiently through the small intestine (ileum), returned via a portal vein to the liver and excreted again into bile (enterohepatic circulation). The cholesterol pool size in the body is subject to feedback regulation not only by cholesterol in a meal but also by bile acid in enterohepatic circulation, and thus hypercholesterolemia therapy is conducted by suppression of re-absorption of bile acid into intestines by using a bile acid adsorbent (anion exchange resin).
The sodium-dependent bile acid transporter is considered to contribute to transport of bile acid. In humans, two isoforms of sodium-dependent bile acid transporter have been identified, and NTCP (Na+/taurocholate cotransporting polypeptide) is expressed mainly in the liver (J. Clin. Invest., 93, 1326-1331, 1994), while ISBT (ileal sodium/bile salt cotransporter) is expressed mainly in the ileum/kidney (J. Biol. Chem., 270, 27228-27234, 1995). With respect to ISBT, direct relationship between a gene mutation accompanied by amino acid substitution and insufficient absorption of bile acid is suggested (J. Clin. Invest., 99, 1880-1887, 1997).
A Na+/H+ exchange transporter (NHE) is a typical cation antiporter, which couples in animal cells with Na+ inflow to discharge H+. NHE is divided into 2 major regions, that is, an amino terminal (N) region containing about 500 amino acids comprising a 10- to 13-times transmembrane region and a carboxyl terminal (C) region comprising about 300 amino acids, and its whole structure is common among isoforms. It is known that the former is an ion transport region comprising an amyloride-binding site, and the latter functions as an activity regulatory region.
As isoforms of NHE in humans, 6 kinds of isoforms i.e. NHE1 to NHE3 and NHE5 to NHE7 are reported. NHE1 is distributed broadly in tissues, and involved in regulation of intracellular pH and cell volume. The activity of NHE1 is promoted by a growth factor or simulation with high osmotic pressure, resulting in an increase in intracellular pH. NHE3 is expressed in the kidney and small intestine, and plays an important role in absorption of Na+. It is thus known that the respective isoforms are different in their expression distribution, regulatory mechanism, and the effect of inhibitor.
NHE1 is considered as one factor increasing intracellular Na+ levels after ischemia and participating in causing myocardial difficulties. It is also reported that the activity of NHE1 in patients with hypertension is significantly higher than in healthy persons. In mice spontaneously developing epilepsy, it is confirmed that the disease is caused by a mutation in NHE (Cell, 91, 139-148, 1997).
P-type ATPase is a membrane enzyme participating in transport of various substrates by utilizing energy upon hydrolysis of ATP. The P-type ATPase is divided into 3 classes, depending on its substrate. Type-1 utilizes heavy metals such as Cu2+ ion and Cd2+ ion as the substrate, possesses an N-terminal characteristic structure involved in binding to heavy metals, and has an 8-times transmembrane structure. Wilson's disease is a disease accompanying an abnormality in Cu2+-ATPase participating in excretion of copper in the liver. Type-2 utilizes alkali metals (K+ ion, Na+ ion), alkaline earth metals (Ca2+ ion) or proton (H+) as the substrate. In particular, H+, K+-ATPase (proton pump) in stomach acid-secreting cells is a target of chemicals such as proton pump inhibitors (omeplazole, lansoprazole etc.) that are therapeutic products for stomach ulcer/duodenum ulcer/reflux esophagitis. Further, Na+, K+-ATPase (sodium pump) is a target of chemicals such as cardiac glycosides used for cardiac diseases, and its activity is inhibited by ouabain. Type-3 is the latest determined type, and utilizes aminophospholipids as the substrate. It is also called aminophospholipid translocase (flippase), and reversely transfer phospholipids selectively from outer to inner layers by using energy generated upon hydrolysis of ATP. It is estimated that uneven distribution of lipids on the biomembrane is thereby maintained. No significant difference in structure is recognized between type-2 and type-3, both of which have a 10-times transmembrane structure (Biochemistry, 34, 15607-15613, 1995; Science, 272, 1495-1497, 1996).
Up to now, 17 isoforms of P-type ATPase of type-3 have been identified in mammals. Among them, FIC1 is expressed in tissues such as the pancreas, small intestine, liver etc., and the relationship between an alteration in its gene and hereditary cholestasis is reported (Nature Genet., 18, 219-224, 1998).
The P-type ATPase of type 3 is considered to play an important role in transport of aminophospholipids and in uneven distribution of lipids on the biomembrane, but the detailed functions and structure of each isoform and the relationship thereof with the disease are not so revealed.
As a pain receptor, a vanilloid receptor subtype 1 (VR1) is a non-selective cation channel with high Ca2+ permeability having outward rectification. It is known that VR1 has a 6-times transmembrane region, possesses an H5 region regarded as forming a pore between fifth and sixth transmembrane sites, and has 3 ankyrin repeat domains at the N-terminal thereof. In addition to VR1 (Biochemical and Biophysical Research Communications, 281, 1183, 2001), VRL (vanilloid receptor-like protein) 1 and VRL2 in humans have been cloned up to now, and have about 40% homology with VR1 respectively (Physiol Genomics 4, 165-174, 2001).
Capsaicin has a vanillyl group and is thus called vanilloid, and is an extraneous ligand of vanilloid receptor. No intrinsic ligand has been revealed. Single electric current measurement revealed that VR1 is activated electrophysiologically directly by capsaicin. Further, VR1 is a receptor of multi-stimuli, which is activated not only by chemical stimulation with capsaicin or the like but also by heat stimulation regarded pain stimulation (at a temperature of higher than 43° C. that is a threshold temperature at which pain is induced in humans) and acid stimulation (tissues are acidified in inflammations and ischemia).
VR1 is activated by stimuli (for example capsaicin, heat, proton) causing pain in the living body, and in a morbid state, these stimuli are considered to occur not singly but simultaneously. Receptiveness of every pain in the living body is not elucidated by only VR1, and the presence of other homologues and cofactors is also estimated. In the previously reported VR family, there are various expression sites and stimulation receptivity, and these are considered to function depending on one another, to transmit pain stimulation.
The sodium-dependent bile acid transporter is considered to play an important role in transport of bile acid in the liver and small intestine, but its detailed mechanism and the relationship thereof with the disease are not so revealed. Full elucidation of the substrate specificity of the sodium-dependent bile acid transporter and its role in bile acid metabolism leads to development of therapeutic products for diseases associated with bile acid metabolism.
As described above, NHE is involved in many morbid states, and elucidation of the mechanism of activation and regulation of each isoform of NHE leads to development of therapeutic products.
Elucidation of detailed functions of P-type ATPase of type 3 leads to development of therapeutic products for diseases such as metabolic diseases, central nerve diseases, genital diseases and cancers associated with P-type ATPase of type 3.
The above-mentioned capsaicin is used as an analgesic for relieving pains in diabetic neurosis and articular rheumatism, and thus elucidation of the structure, function and mutual relationship of VR family is considered to lead to development of therapeutic products for pains as a whole.